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He cell population spreads across the substrate can be calculated. A common approach to quantify the cell migration rate in a barrier assay is to report the percentage change in area [15,16,18?20]. This can be expressed as M(t) A(t){A(0) |100, A(0) ??where A(0) is the initial area enclosed by the population of cells, A(t) is the area enclosed by the population of cells at time t, and M(t) is the percentage change in area at time t. Estimates of cell migration rates using equation (1) are often obtained by hand tracing the area enclosing the spreading cell population on an image of the assay [21,22]. Unfortunately, hand tracing the area enclosed by the leading edge of a spreading cell population is subjective [23]. To overcome this limitation, automated image analysis software, including ImageJ [24] and MATLAB’s Image Processing Title Loaded From File Toolbox [25], have become important alternatives to hand tracing [8,26]. These software tools use edge detection and segmentation algorithms to determine the location of the leading edge of the spreading cell population. This data can then be used to quantify the cell migration rate inSensitivity of Edge Detection Methodsterms of equation (1). In addition to using automatic edge detection algorithms, it is also possible to implement user-defined edge detection options in MATLAB’s Image Processing 16985061 Toolbox [25] so that the user has complete control over the choice of image detection thresholds. Since there is no standardized method for quantifying the location of the leading edge in a barrier assay, it is often difficult, if not impossible, to meaningfully compare published measures of cell migration in terms of equation (1). This difficulty is exacerbated by the fact that previously published results have been obtained using different image analysis techniques and the details are not always reported [27?1]. To address this limitation, here we apply three different edge detection techniques to a set of images from a two-dimensional barrier assay describing the collective spreading of a population of 3T3 fibroblast cells. We apply three different edge detection techniques to the same experimental data set and compare results from two commonly used automatic edge detection techniques and one manual edge detection technique. Our results indicate that the location of the leading edge is sensitive to the details of the edge detection procedure and this can lead to significantly different quantitative estimates of cell migration. Using a reasonable range of threshold values we show that estimates of cell migration, given by equation (1), can vary by as much as 25 for the same data set. To provide further insight into the edge detection techniques, we also interpret our results using a mathematical model to quantitatively describe the temporal cell spreading process associated with the barrier assay. Using previously-determined estimates of the cell diffusivity [17], we show that the location of the leading edge, as defined by the image detection methods, corresponds to contours of cell Title Loaded From File density in the range of approximately 1? of the maximum cell packing density. Comparing the location of the leading edge determined by the image detection methods and the mathematical model of the cell spreading provides us with a simple, but meaningful, physical interpretation of the threshold parameters used in the image detection methods.with serum free medium (SFM; culture medium without FCS) and replaced with 0.5 mL of culture medi.He cell population spreads across the substrate can be calculated. A common approach to quantify the cell migration rate in a barrier assay is to report the percentage change in area [15,16,18?20]. This can be expressed as M(t) A(t){A(0) |100, A(0) ??where A(0) is the initial area enclosed by the population of cells, A(t) is the area enclosed by the population of cells at time t, and M(t) is the percentage change in area at time t. Estimates of cell migration rates using equation (1) are often obtained by hand tracing the area enclosing the spreading cell population on an image of the assay [21,22]. Unfortunately, hand tracing the area enclosed by the leading edge of a spreading cell population is subjective [23]. To overcome this limitation, automated image analysis software, including ImageJ [24] and MATLAB’s Image Processing Toolbox [25], have become important alternatives to hand tracing [8,26]. These software tools use edge detection and segmentation algorithms to determine the location of the leading edge of the spreading cell population. This data can then be used to quantify the cell migration rate inSensitivity of Edge Detection Methodsterms of equation (1). In addition to using automatic edge detection algorithms, it is also possible to implement user-defined edge detection options in MATLAB’s Image Processing 16985061 Toolbox [25] so that the user has complete control over the choice of image detection thresholds. Since there is no standardized method for quantifying the location of the leading edge in a barrier assay, it is often difficult, if not impossible, to meaningfully compare published measures of cell migration in terms of equation (1). This difficulty is exacerbated by the fact that previously published results have been obtained using different image analysis techniques and the details are not always reported [27?1]. To address this limitation, here we apply three different edge detection techniques to a set of images from a two-dimensional barrier assay describing the collective spreading of a population of 3T3 fibroblast cells. We apply three different edge detection techniques to the same experimental data set and compare results from two commonly used automatic edge detection techniques and one manual edge detection technique. Our results indicate that the location of the leading edge is sensitive to the details of the edge detection procedure and this can lead to significantly different quantitative estimates of cell migration. Using a reasonable range of threshold values we show that estimates of cell migration, given by equation (1), can vary by as much as 25 for the same data set. To provide further insight into the edge detection techniques, we also interpret our results using a mathematical model to quantitatively describe the temporal cell spreading process associated with the barrier assay. Using previously-determined estimates of the cell diffusivity [17], we show that the location of the leading edge, as defined by the image detection methods, corresponds to contours of cell density in the range of approximately 1? of the maximum cell packing density. Comparing the location of the leading edge determined by the image detection methods and the mathematical model of the cell spreading provides us with a simple, but meaningful, physical interpretation of the threshold parameters used in the image detection methods.with serum free medium (SFM; culture medium without FCS) and replaced with 0.5 mL of culture medi.